Dengue serotype 1 vaccine development: The live attenuated DEN1 vaccine candidate virus rDEN1del30 has been evaluated in Phase I clinical trials and was found to be safe and immunogenic at a dose of 1000 PFU. It is a promising vaccine candidate for inclusion in a tetravalent dengue vaccine formulation. Additional studies have been fully enrolled to determine the number of doses and the timing of the doses needed to achieve optimal immunization. The studies involve two doses of vaccine given at an interval of 4 or 6 months. The results will be fully reported pending complete analysis, but preliminary analysis indicates that the first dose is highly immunogenic but the second dose is poorly infectious at 6 months. A separate clinical trial to determine the 50% infectious dose (HID50) was initiated this year and preliminary results indicate that at the lowest dose administered (10 PFU), the vaccine remains fully infectious. Dengue serotype 2 vaccine development: rDEN2/4del30(ME) is an attenuated chimeric dengue virus in which the prM and E structural proteins of the DEN4 candidate vaccine, rDEN4del30, have been replaced by those of the prototypic DEN2 NGC virus. The live attenuated DEN2 vaccine candidate virus rDEN2/4del30(ME) has been evaluated in Phase I clinical trials and found to be safe and immunogenic at a dose of 1000 PFU. It is a promising vaccine candidate for inclusion in a tetravalent dengue vaccine formulation. Additional studies were initiated to address the timing of two doses of this vaccine candidate. Results obtained this year showed that a second dose administered at 6 months did not boost the immune response to the primary dose, presumably due to complete neutralization of the second dose by the existing antibody. A clinical trial to determine the 50% infectious dose (HID50) was initiated this year and preliminary results indicate that at the lowest dose administered (10 PFU), approximately half of the volunteers seroconverted to DEN2, indicating that the HID50 is close to 10 PFU. Dengue serotype 3 vaccine development: rDEN3/4del30(ME) is an attenuated chimeric dengue virus in which the prM and E structural proteins of the DEN4 candidate vaccine rDEN430 have been replaced by those of a DEN3 wild type virus. rDEN3/4del30(ME) or placebo was previously evaluated at a dose of 1,000 or 100,000 PFU in 28 healthy dengue-naive adult volunteers per dose. Less than 40% of vaccinees were infected with either dose indicating this vaccine candidate is not sufficiently infectious to be used as a component of a tetravalent dengue vaccine formulation. Studies were initiated with two additional DEN3 vaccine candidates in which 1) the 3 prime UTR (untranslated region) of rDEN3 was replaced with that derived from attenuated rDEN4del30 to generate rDEN3-3D4del30, and 2) a pair of 3 prime UTR deletions were introduced into DEN3 to create vaccine candidate rDEN3del30/31. A clinical trial completed this year showed that the rDEN3-3D4del30 vaccine was well-tolerated and 80% of the volunteers were infected. In a separate clinical trial completed this year, the rDEN3del30/31 vaccine was shown to be well-tolerated with 95% of the volunteers becoming infected. Both of these new DEN3 vaccine candidates are promising candidates for inclusion in a tetravalent vaccine formulation. Dengue serotype 4 vaccine development: We currently consider the rDEN4del30 vaccine our leading DEN4 candidate for inclusion in the tetravalent vaccine. However, rDEN4del30-200,201 is a live attenuated DENV-4 vaccine candidate specifically designed to further attenuate the rDEN4del30 parent virus. In a previous study, 5 of 20 vaccinees who received rDEN4del30 at 100,000 PFU developed moderately elevated levels of serum alanine aminotransferase (ALT). Mutational analysis of DEN4 revealed that the 200,201 mutation of NS5 greatly restricted virus replication in human hepatocarcinoma HuH-7 cells. In pre-clinical animal studies, the vaccine candidate rDEN4del30-200,201 was shown to be more attenuated than rDEN4del30 in both SCID-HuH-7 mice and rhesus macaques. In a recent clinical study, the vaccine infected 95% of vaccinees and was well tolerated without inducing ALT elevations. Although virus was not recovered from the serum of any vaccinee, moderate levels of neutralizing antibody were induced. The rDEN4del30-200,201 vaccine candidate is considered a back up candidate for inclusion in a tetravalent formulation should the rDEN4del30 vaccine candidate display an unacceptable dominance in the tetravalent formulation. A second back up candidate is rDEN4del30-4995, which is a DEN4 vaccine candidate specifically designed as a further attenuated derivative of the rDEN4del30 parent virus. In the recent clinical study, 28 healthy adult volunteers were randomized to receive 100,000 PFU of rDEN4del30-4995 (20) or placebo (8) as a single subcutaneous injection. The vaccine was safe, well-tolerated, and immunogenic. An asymptomatic generalized maculopapular rash and elevations in ALT levels were observed in 10% of the rDEN4del30-4995 vaccinees. None of the volunteers became viremic, yet 95% developed a four-fold rise in neutralizing antibody titers. Thus rDEN4del30-4995 was demonstrated to be safe, highly attenuated, and immunogenic. Unfortunately, an asymptomatic localized erythematous rash at the injection site was seen in 17 of 20 rDEN4del30-4995 vaccinees and this virus will not be studied further. Tetravalent studies: Clinical studies were initiated this year to evaluate three different tetravalent formulations containing 1000 PFU of each individual vaccine candidate. Each formulation contains rDEN1del30 and rDEN2/4del30 along with the following DEN3 and DEN4 vaccine candidates: TV001 contains rDEN3-3D4del30 and rDEN4del30, TV002 contains rDEN3-3D4del30 and rDEN4del30-200,201, and TV003 contains rDEN3del30/31 and rDEN4del30. Studies with all three formulations are ongoing in cohorts of 20 vaccinees and 8 placebo recipients. West Nile virus vaccine studies: Clinical studies are ongoing with a live attenuated West Nile virus vaccine candidate, WNVDEN4del30. Studies at 1000 and 100,000 infectious units per vaccinee indicated the vaccine was safe with low viremia, but was immunogenic in only 80% of vaccinees. To achieve a higher take rate, a study is ongoing in which two doses of 100,000 infectious units are given six months apart. The study is fully enrolled, and preliminary analysis indicates that the vaccine given as two doses is safe and immunogenic.